Abstract Neonatal thymic-derived Foxp3+ T regulatory cells (tTregs) are required for the development of immune homeostasis and limiting organ specific autoimmune disease. The molecular details of TCR-pMHC interactions, and the specific downstream signaling pathways that allow neonatal tTregs to develop, seed peripheral tissues and regulate acute inflammation are not well understood. We hypothesize that a subset of neonatal tTregs distinguishes health from disease via the expression of TCR with specificity for self-ligands that are upregulated during inflammatory conditions. This tTreg TCR recognition property manifests as graded levels of immune suppression based on the context and magnitude of the inflammatory setting. Preliminary data further suggest that the development of these tTreg clones within the neonatal selection window is temporally constrained by negative selection, and is predicated on kinetic proofreading, with TCR-self-pMHC dwell times within a conventional binding mode as a key to specifying tTreg development. To test our hypothesis, we will first identify endogenous self-ligands recognized by Foxp3+ CD4 tTreg cell subsets. Our approach is based on our proven ability to identify self-ligands recognized by T cells, paired with mass spectrometry of MHC-II bound self-peptides presented on APC isolated from different anatomical locations, as well as high-throughput pipelines for determining recognition properties of individual T cell clonotypes. Using paired sets of TCR-self-pMHC combinations our second aim will directly examine whether neonatal tTreg selection is based the dwell time of the interaction, and assess the influence of ?unconventional? TCR/self- pMHC binding modes in selecting the neonatal tTreg repertoire. Aim 3 will identify synergies between self- pMHC presentation by thymic APCs and the quality of TCR signals generated by thymocytes that define the neonatal tTreg selection window. Signaling by the Tec family kinase, Itk, is proposed to regulate a signaling threshold that separates Foxp3 Treg selection from late stage deletion by amplifying pro-survival TCR signals derived from moderate dwell-time ligands via NFAT and NF-?B signaling pathways. Finally, in mature tTregs, we propose that Itk functions to amplify weak TCR responses, thereby allowing mature Tregs to recognize gradients in self-antigen displayed. These studies will provide important insights into the fine-tuning of T cell responses and the signaling pathways that discriminate effector cells from regulatory cells, leading to rational approaches in the design of therapeutics to manipulate immune responses for treatments of cancer and autoimmune diseases.